Effect of AGTR1 A1166C genetic polymorphism on coronary artery lesions and mortality in patients with acute myocardial infarction

The pathogenesis and prognosis of patients with acute myocardial infarction (AMI) may be influenced by both genetic and environmental factors. Findings on the relationship of polymorphisms in various genes encoding the renin-angiotensin-aldosterone system with coronary artery lesions and mortality in AMI patients are inconsistent. The aim of this study was to determine whether the AGTR1 A1166C genetic polymorphism affects coronary artery lesions and 1-year mortality in post-AMI patients. Patients with their first AMI admitted to Cho Ray Hospital, Vietnam, from January 2020 to August 2021 were enrolled in this prospective clinical study. All participants underwent invasive coronary angiography and were identified as having the genotypes of AGTR1 A1166C by way of a polymerase chain reaction method. All patients were followed up for all-cause mortality 12 months after AMI. The association of the AGTR1 A1166C polymorphism with coronary artery lesions and 1-year mortality was evaluated using logistic regression and Cox regression analysis, respectively. Five hundred and thirty-one AMI patients were recruited. The mean age was 63.9 ± 11.6 years, and 71.6% of the patients were male. There were no significant differences in the location and number of diseased coronary artery branches between the AA and AC+CC genotypes. The AC and CC genotypes were independently associated with ≥ 90% diameter stenosis of the left anterior descending (LAD) artery (odds ratio = 1.940; 95% confidence interval (CI): 1.059–3.552, p = 0.032). The 1-year all-cause mortality rate difference between patients with the AC and CC genotypes versus those with the AA genotype was not statistically significant (hazard ratio = 1.000, 95% CI: 0.429–2.328, p = 1.000). The AGTR1 A1166C genetic polymorphism is associated with very severe luminal stenosis of the LAD but not with mortality in AMI patients.


Background
The pathogenesis and prognosis of patients with acute myocardial infarction (AMI) may be influenced by both genetic and environmental factors.Findings on the relationship of polymorphisms in genes encoding the renin-angiotensin-aldosterone system with coronary artery lesions and mortality in AMI patients are inconsistent.The aim of this study was to determine whether the AGTR1 A1166C genetic polymorphism affects coronary artery lesions and 1-year mortality in post-AMI patients.

Methods
Patients with their first AMI admitted to Cho Ray Hospital, Vietnam, from January 2020 to August 2021 were enrolled in this prospective clinical study.All participants underwent invasive coronary angiography and were identified as having the genotypes of AGTR1 A1166C by way of a polymerase chain reaction method.All patients were followed up for all-cause mortality 12 months after AMI.The association of the AGTR1 A1166C polymorphism with coronary artery lesions and 1-year mortality were evaluated using logistic regression and Cox regression analysis, respectively.Results 531 AMI patients were recruited.The mean age was 63.9 ± 11.6 years, and 71.6% of the patients were male.There were no significant differences in the location and number of diseased coronary artery branches between the AA and AC+CC genotypes.The AC and CC genotypes were independently associated with ≥ 90% diameter stenosis of the left anterior descending (LAD) artery (odds ratio = 1.940; 95% confidence interval (CI): 1.059 -3.552, p = 0.032).The difference in the 1-year allcause mortality rate between patients with the AC and CC genotypes versus those with the AA genotype was not statistically significant (hazard ratio = 1.000, 95% CI: 0.429-2.328,p = 1.000).

Conclusions
The AGTR1 A1166C genetic polymorphism is associated with very severe luminal stenosis of the LAD, but not with mortality in AMI patients.This statement is required for submission and will appear in the published article if the submission is accepted.Please make sure it is accurate.

Abstract
Background: The pathogenesis and prognosis of patients with acute myocardial infarction (AMI) may be influenced by both genetic and environmental factors.Findings on the relationship of polymorphisms in genes encoding the renin-angiotensin-aldosterone system with coronary artery lesions and mortality in AMI patients are inconsistent.The aim of this study was to determine whether the AGTR1 A1166C genetic polymorphism affects coronary artery lesions and 1-year mortality in post-AMI patients.
Methods: Patients with their first AMI admitted to Cho Ray Hospital, Vietnam, from January 2020 to August 2021 were enrolled in this prospective clinical study.All participants underwent invasive coronary angiography and were identified as having the genotypes of AGTR1 A1166C by way of a polymerase chain reaction method.All patients were followed up for all-cause mortality 12 months after AMI.The association of the AGTR1 A1166C polymorphism with coronary artery lesions and 1-year mortality were evaluated using logistic regression and Cox regression analysis, respectively.
Results: 531 AMI patients were recruited.The mean age was 63.9 ± 11.6 years, and 71.6% of the patients were male.There were no significant differences in the location and number of diseased coronary artery branches between the AA and AC+CC genotypes.The AC and CC genotypes were independently associated with ≥ 90% diameter stenosis of the left anterior descending (LAD) artery (odds ratio = 1.940; 95% confidence interval (CI): 1.059 -3.552, p = 0.032).The difference in the 1-year all-cause mortality rate between patients with the AC and CC genotypes versus those with the AA genotype was not statistically significant (hazard ratio = 1.000, 95% CI: 0.429-2.328,p = 1.000).
Conclusions: The AGTR1 A1166C genetic polymorphism is associated with very severe luminal stenosis of the LAD, but not with mortality in AMI patients.

Coronary artery disease (CAD) is the predominant cause of death around the globe(1).
Acute myocardial infarction (AMI) is the most severe clinical manifestation of CAD.The essential mechanism of AMI is the rupture or erosion of atherosclerotic plaques leading to thrombus formation causing complete or incomplete occlusion of epicardial coronary artery branches (2).In clinical practice, AMI is a common cardiovascular emergency and requires aggressive management.Although the survival status of patients with AMI has improved in recent decades, the burden of mortality and morbidity remains significant.The 1-year death rate in patients after AMI ranges from 10 to 12%(3).
Coronary artery lesions and mortality in AMI patients can be caused by both environmental and genetic factors.Many genetic markers associated with AMI have been identified, including genes encoding components of the renin-angiotensin-aldosterone system (RAAS).The AGTR1 gene encoding the angiotensin II receptor type 1 (AT1) is located on the long arm of chromosome 3 (3q21-25), and A1166C is the most studied polymorphism(4).This gene variant is at the untranslated 3' region and has a base substitution of adenine with cytosine at nucleotide position 1166 of the mRNA sequence.
In patients carrying the AGTR1 A1166C genetic polymorphism, there is an increased expression of the AT1 receptor (5).Angiotensin II, the main effector in RAAS, acts on the AT1 receptor and causes vasoconstriction, aldosterone secretion, cell proliferation, vascular remodeling, endothelial dysfunction, and atherosclerosis (6,7).
The AGTR1 A1166C genetic variant has been shown in previous studies to be associated with the risk of AMI.A meta-analysis comprising of 18 case-control studies demonstrated that the C allele is a risk factor for AMI (8).However, few studies have investigated the association between this genetic polymorphism and coronary artery lesions and mortality in patients with AMI.Kruzliak P et al. found that the CC genotype is associated with a higher risk of three-vessel stenosis and also a higher proportion of left anterior descending (LAD) artery infarction (9).In contrast, Araujo MA et al. did not detect a relationship between the AGTR1 A1166C variant and coronary artery injuries when comparing the CC Acute myocardial infarction (AMI) AT1R (rs5186) genotype with AC and AA genotypes in AMI patients (10).However, AGTR1 A1166C has been revealed in several studies to be a prognostic factor for mortality in patients with AMI (9,11,12), although other studies have not found any predictive value (13,14).Given the lack of data and the conflicting results between various studies, we conducted this study with the aim of investigating the effect of the AGTR1 A1166C genetic variant on coronary artery lesion characteristics and mortality in Vietnamese patients with AMI.

Study population
In this prospective clinical study, we enrolled patients who were admitted to the

Study protocol
We collected data on participants' clinical characteristics, including age, sex, clinical type of AMI, and Killip class.Traditional cardiovascular risk factors were also collected, namely hypertension (according to ESC/ESH guidelines) (15), diabetes mellitus (according to ADA guidelines) (16), dyslipidemia (according to NCEP ATP-III guidelines) (17), obesity (according to the WHO classification for the Asian population) (18), smoking, and a family history of premature CAD.Laboratory parameters, such as Troponin I concentration, estimated glomerular filtration rate (eGFR), and left ventricular ejection fraction (LVEF) were also recorded.The LVEF of patients was assessed using Simpson's method for echocardiography.All patients underwent invasive coronary angiography.The characteristics of coronary artery lesions were noted, i.e., the position of diseased coronary artery branches with ≥ 50% stenosis of the diameter, the number of diseased coronary artery branches, and very severe stenosis of the artery coronary artery branches (≥ 70% stenosis of the left main and ≥ 90% stenosis of the other epicardial coronary arteries).Two milliliters of venous blood were drawn from each patient for genotyping of the AGTR1 A1166C polymorphism.
The patients were followed up during their hospital stay and up to 12 months from the date of admission.The primary outcome measured was all-cause mortality.Post-discharge follow-up was conducted through revisits and hospitalization at Cho Ray Hospital or telephone interviews.

Genotyping of AGTR1 A1166C
Genomic deoxyribonucleic acid (DNA) for all participants was extracted from peripheral leukocytes using the GeneJet TM Whole Blood Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol.
For each sample, AGTR1 A1166C was genotyped by two separate polymerase chain reactions (PCR).Each reaction used three primers with similar melting temperatures to determine the presence of allele A or allele C based on the appearance of a 342-basepair DNA band on electrophoresis.All the PCRs were performed using a SimpliAmp TM Thermal Cycler (Thermo Fisher Scientific).The details of the PCR primers, components, and conditions are described in the Supplementary materials.
Twenty DNA samples were randomly chosen and directly sequenced using appropriate primers and conditions in an ABI 3500 sequencer (Thermo Fisher Scientific).The protocol for direct sequencing has been described previously (19)(20)(21).The DNA samples with an identified genotype were used as positive controls for PCR.

Statistical analysis
Qualitative variables were presented as frequencies and percentages.The Kolmogorov-Smirnov test was used to check the normal distribution of quantitative variables, which were expressed as mean with standard deviation (SD) if normally distributed or median with interquartile range [IQR] if not normally distributed.Consistency with the Hardy-Weinberg equilibrium of genotypes was assessed using Chi-squared tests.Differences in the characteristics of coronary artery lesions between genotypes of the AGTR1 A1166C genetic polymorphism were evaluated using Chi-squared or Fisher's exact tests.Factors affecting the degree of ≥ 90% stenosis of the LAD diameter were assessed by odds ratio (OR) and 95% CI in univariate and multivariable logistic regression analyses.Differences in the genotypes of the AGTR1 A1166C polymorphism and other factors between those who survived and those who did not were compared using Chi-squared or Fisher's exact tests for qualitative variables and Student's t-test or the Mann-Whitney U test based on the data distribution.Factors associated with 1-year all-cause mortality in the univariate analysis were analyzed in a multivariable Cox regression model to identify independent prognostic factors.
Statistical analysis was performed using SPSS Statistics for Windows version 22.0 (IBM Corp., Armonk, NY, USA).A p-value of less than 0.05 was considered to be statistically significant.

Characteristics of the study population
This study included 531 patients with their first AMI, most of whom were male (71.6%) (Table 1).The mean age of the participants was 63.9 ± 11.6 years.ST-segment elevation AMI was diagnosed in 63.3% of patients.Regarding the degree of acute heart failure, the proportion of patients with Killip class ≥ II was 23.5%.The prevalent diseased coronary artery was the LAD (89.1%).Three-vessel disease was predominant (40.7%) on coronary angiography.The majority of patients underwent coronary revascularization (94.7%) with fibrinolysis, percutaneous coronary intervention, or coronary bypass surgery.In addition, participants received standard medical therapy with aspirin (99.8%),P2Y12 inhibitor (100.0%),statin (98.5%),ACEI/ARB (90.4%), and beta-blocker (76.5%).
The genotypes of the AGTR1 A1166C polymorphism in our study were not in agreement with the Hardy-Weinberg equilibrium (p = 0.01) (Table 2).The AA genotype was most common in AMI patients (89.7%).In terms of traditional cardiovascular risk factors, dyslipidemia was the most frequent factor (89.6%), and a family history of premature CAD was not common (6.6%).

Association of AGTR1 A1166C genetic polymorphism with coronary artery lesions in AMI patients
There was no association between the AGTR1 A1166C genetic variant and the location of diseased coronary artery branches, i.e., LM, LAD, LCx, and RCA (Table 3).No significant differences in the number of diseased coronary artery branches were found between patients carrying the AA genotype and those with the AC and CC genotype.However, the univariate analysis showed that patients with the AC and CC genotypes had a greater risk of ≥ 90% LAD stenosis than those with the AA genotype (p = 0.028).Univariate logistic regression analysis also showed that obesity and left ventricular ejection fraction were related to very severe stenosis of the LAD (p = 0.022 and p < 0.001, respectively).After adjusting for confounding factors in the multivariable logistic regression analysis, the AC and CC genotype was found to be independently associated with very severe stenosis of the LAD in AMI patients (OR = 1.940; 95% CI: 1.059 -3.552; p = 0.032) (Table 4).

Association of the AGTR1 A1166C genetic polymorphism and other factors with 1-year all-cause mortality in AMI patients
There were 58 all-cause deaths during the 1-year follow-up (10.9%), including 52 patients with the AA genotype and 6 patients with the AC or CC genotype (Table 5).In univariate analysis, diabetes mellitus, age, Killip class ≥ II, Troponin I on admission, eGFR, LVEF, and ACEI/ARB therapy were found to be associated with 1-year all-cause mortality.

Discussion
The genotypes of the AGTR1 A1166C polymorphism in our study were not in agreement with the Hardy-Weinberg equilibrium, possibly because our study population was a group of patients with AMI, not the entire population of case and control groups as in case-control studies.We did not select a control group because doing so was not necessary to answer the research question of whether the AGTR1 A1166C variant is associated with coronary artery lesions and mortality in AMI patients.The CC genotype was the least common (1.1%) in our study population.This feature is consistent with other studies on different races, not just on Asian populations (9,10,(22)(23)(24).
Our study showed that patients with the AC and CC genotypes had a higher rate of ≥ LAD 90% stenosis than AA genotype carriers.The study by Kruzliak P et al. demonstrated that acute coronary syndrome patients carrying the CC genotype in the Slovak Republic had a 4.08-fold higher risk of LAD infarction and a 3.87 times higher risk of three-vessel disease compared with those having the AA and AC genotypes (9).Nevertheless, the study by Araujo MA et al. found no differences in the number of diseased coronary vessels and morphological features of the atherosclerotic plaque among the AGTR1 A1166C genotypes (10).The results of other studies are conflicting regarding the effect of AGTR1 A1166C variants on coronary artery lesions due to differences in genotype distributions across countries and races, as well as the frequency of environmental factors affecting coronary atherosclerosis.In terms of pathogenesis, the AT1 receptor may be overexpressed in carriers of the AGTR1 A1166C variant, leading to increased adverse effects of angiotensin II on coronary atherosclerosis.Angiotensin II promotes coronary artery injury possibly through various pathophysiological phenomena, such as modulation of the inflammatory response, stimulation of the production of various cytokines, such as IL-6, TNF-α, and COX-2, and promotion of the generation of reactive oxygen species, enhanced oxidative stress, decreased nitric oxide production, and increased endothelial dysfunction(3).
The AGTR1 A1166C genetic polymorphism was not found to be associated with 1-year allcause mortality in AMI patients in the present study.Similar results have been observed in other studies (13,14).The CC genotype was not found to be associated with in-hospital mortality in the GEMIG (Genetics and Epidemiology of Acute Myocardial Infarction in the Greek Population) study in Greece (14).A study by Brscic E et al. involving young Italian patients with AMI concluded that the AGTR1 A1166C polymorphism was not related to the composite end points of major cardiovascular events, including cardiovascular mortality, myocardial infarction, and revascularization procedures during the follow-up period of 46 ± 12 months (13).
In contrast, the AGTR1 A1166C genetic polymorphism has been shown in several studies to be a predictor of mortality in AMI patients (9,11,12).Kruzliak P et al. found that the CC genotype is associated with a 6.48-fold increased risk of sudden cardiac death within 24 hours of emergency room admission in patients with acute coronary syndromes (9).After adjusting for the cause of death, the CC genotype was found in a large prospective study in France to be an independent predictor of post-AMI cardiovascular mortality during follow-up (median 2.5 years) (11).In addition, a study by Franco E et al. concluded that the AC genotype was associated with death, new myocardial infarction, and coronary revascularization in young Italian patients with AMI during the follow-up period of 9 ± 4 years (12).
The inconsistency in the results of various studies on the influence of the AGTR1 A1166C genetic variant on mortality in AMI patients could be due to differences in the occurrence of the AGRT1 A1166C genotypes across distinct ethnicities, countries, and geographic regions, as well as differences in study populations, study designs, and survival follow-up periods.In addition, ACEI and ARB have been shown in previous studies to improve mortality in patients after AMI (25)(26)(27).Therefore, the impact of ACEI and ARB might obscure the predictive value of the AGTR1 A1166C polymorphism for mortality.The proportion of patients using ACEI/ARB in our study was very high (90.4%).
Our study had some limitations that should be taken into consideration.First, this study was conducted in only one center, so it might not be representative of the genotype characteristics of the AGTR1 A1166C variant in Vietnam.Next, the pathophysiology of AMI includes the interaction of many factors and genes, so the AGTR1 A1166C might not fully explain the association of this polymorphism with coronary artery lesions and mortality in patients with AMI.Finally, the study was observational and thus did not intervene in treatment strategies and adherence to therapy in AMI patients.

Conclusions
The AGTR1 A1166C genetic polymorphism is associated with very severe luminal stenosis of the LAD.Nevertheless, it is not associated with 1-year all-cause mortality in Vietnamese patients after AMI.These results contribute to the genetic assessment of coronary artery lesions, but further studies with larger sample sizes and longer follow-up time are needed to consider the prognostic role of the AGTR1 A1166C variant in patients with AMI.
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